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Abstract

Introduction

The ability to degrade neutrophil extracellular traps (NETs) is reduced in a subset
of patients with systemic lupus erythematosus (SLE). NETs consist of chromatin covered
with antimicrobial enzymes and are normally degraded by DNase-I, an enzyme which is
known to have reduced activity in SLE. Decreased ability to degrade NETs is associated
with disease activity. In the current study we investigated how the ability of serum
from SLE patients to degrade NETs varies during the course of SLE as well as what
impact this may have for the clinical phenotype of SLE.

Methods

Serum from 69 patients with SLE, included in a prospective study, was taken every
60 days for a median of 784 days. The ability of serum to degrade NETs was determined
and associated with clinical parameters occurring before and at the time of sampling,
as well as after sampling by using conditional logistic regression.

Results

As many as 41% of all patients in the study showed decreased ability to degrade NETs
at least once, but with a median of 20% of all time points. Decreased degradation
was associated with manifestations of glomerulonephritis as well as low complement
levels and elevated levels of antibodies directed against histones and DNA. Furthermore,
the odds ratio for the patient to develop alopecia and fever after an episode of decreased
NETs degradation was increased by four to five times compared to normal.

Conclusions

Decreased degradation of NETs is associated with clinical manifestations in SLE and
may contribute to disease pathogenesis. Potential therapeutics restoring the ability
to degrade NETs could be beneficial for certain patients with SLE.

Keywords:

Introduction

The autoimmune disease systemic lupus erythematosus (SLE) is a complex and heterogeneous
disease with the patients displaying a variety of symptoms ranging from glomerulonephritis
to skin rashes and chronic fatigue. A common feature of SLE is the generation of anti-nuclear
antibodies. It has been hypothesized that SLE evolves from the inefficient or improper
clearance and degradation of dying cells [1-4]. Numerous genes have been associated with the disease, spanning from immune modulatory
genes to complement factors [5], all crucial to ensure a proper immune response and efficient clearance of apoptotic
and necrotic cells. In 2004, a new potential antigen source in SLE was discovered
with the description of neutrophil extracellular traps (NETs) [6]. NETs consist of chromatin and antimicrobial enzymes that are released from neutrophils
as a "last-resort" defense to trap and kill pathogens. It was subsequently shown in
two independent studies that NETs are efficiently degraded in serum from healthy controls,
whereas this ability is reduced in a subpopulation of SLE patients [7,8]. The patients with decreased ability to degrade NETs suffered from a severe form
of SLE with glomerulonephritis and additionally exhibited autoantibodies that recognized
NETs. Numerous recent reports further show involvement of NETs in SLE. This spans
from how NETs are more easily formed by neutrophils isolated from SLE patients, potentially
through elevated interferon-α levels or the presence of activating antibodies in these
patients to how non-degradable complexes of chromatin and antimicrobial peptides are
found in SLE sera [9]. Together, this all could contribute to the tissue damage in SLE [10]. It has long been known that SLE patients display a decreased ability to degrade
DNA [11] and there are many theories why this is the case. DNase-I is the enzyme responsible
for degradation of NETs and it is inhibited by globular actin. Actin may be released
by platelets, and dying cells during inflammation [12] and has also been shown to prevent excessive chromatin degradation in apoptotic and
necrotic cells [13]. Further, autoantibodies against DNA could shield the DNA from DNase-I and have additionally
been described to cross react directly with the enzyme potentially leading to inhibition
[14]. We also showed that C1q binds to NETs and prevents degradation [8], indicating formation of non-degradable complexes on NETs consisting of autoantibodies
and complement. Interestingly, in our previous study we observed that the decreased
ability of serum from SLE patients to degrade NETs is mostly not permanent but changes
between time points with different disease activity [8]. To thoroughly determine the extent of this phenomenon, we used serum samples from
a prospective study where 69 patients with SLE were followed for up to five years
with samples taken approximately every two months. At each sampling, we measured the
ability of patient serum to degrade NETs. Clinical manifestations, laboratory variables
and treatments were registered at all time-points in the patients and these variables
were used to determine temporal associations with decreased ability to degrade NETs.
We identified a number of distinct clinical manifestations and laboratory variables
that preceded the time-point of decreased NET degradation and some that appeared at
the same time-point as well as after the time-point of decreased NET degradation.

Materials and methods

Patients and sample preparation

Sixty nine patients (6 males and 63 females) with a majority of Caucasian origin,
except for two with Asian and one of Hispanic origin, with a median age of 39 (range
18 to 76) with established SLE, fulfilling at least four or more American College
of Rheumatology (ACR) 1982 classification criteria for SLE [15] were recruited to this prospective study at the Department of Rheumatology, Skåne
University Hospital in Lund (Sweden). The distribution of ACR classification criteria
for SLE of the patients is described in Table 1. The overall aims of the prospective follow-up program were partly to improve care
by close monitoring of SLE-patients at risk of complications and partly to identify
risk factors, clinical and laboratory variables, for disease exacerbations. Disease
activity was recorded at every visit using the SLE Disease Activity Index 2000 (SLEDAI-2K)
[16] together with infections and other clinically relevant information. Visits were scheduled
every two months with a range of 46 to 74 days, depending on disease activity. The
median study duration for the patients was 784 days (mean 921 days) with a range of
139 to 1,792 days corresponding to a median of 14 (2 to 43) visits. Only visits within
46 to 74 days were used for temporal associations. Sera from 77 healthy volunteers
(15 males and 62 females) with a median age of 42 (range 19 to 77) were used as controls.
All patients and healthy controls gave informed consent to participate in the study,
which was approved by the local ethics committee (Lund University) according to the
Helsinki declaration.

Isolation of neutrophils

Neutrophils were isolated from healthy volunteers according to a previously published
method [17]. Briefly, blood from a healthy volunteer was separated by centrifugation on a Histopaque
1119 column (Sigma-Aldrich, St Louise, MO, USA), the granulocyte-rich fraction was
isolated, washed and neutrophils were isolated by centrifugation on a Percoll gradient
(85 to 65%) (GE Healthcare Biosciences, Uppsala, Sweden) and isolated from the intersection
of the 75% and 70% layers, washed and resuspended in RPMI with 10 mM Hepes at 1 ×
106 cells/ml. Purity of neutrophils (>80%) was determined by forward-side scatter analysis
in combination with surface marker expression defined as CD14low/CD15+/CD16+ by PE-labeled anti-CD14 (BD), FITC-labeled anti-CD15 and APC-labeled anti-CD16 (both
from Immunotools, Friesoythe, Germany) in a CyFlow Space (Partec, Münster, Germany)

Generation and degradation of NETs

Freshly isolated neutrophils from healthy volunteers, 50,000/sample, were seeded onto
a 96-well flat bottom plate (Nunc, Thermo Fisher Scientific Inc, Waltham, MA, USA)
with 20 nM PMA (Sigma-Aldrich) for 4 h at 37°C and 5% CO2 to generate NETs. After incubation, cell medium was removed and 10% patient sera,
control sera or sera with 5 to 320 μg/ml hydroxychloroquine (Sigma-Aldrich) in 10
mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2 and 2 mM CaCl2 were added to NETs and incubated for 60 minutes at 37°C. During this time, degraded
NETs were released into solution. Aliquot of the solution was then transferred to
PBS with a final concentration of 2 mM EDTA to stop further degradation and DNA content
was quantified using picoGreen (Invitrogen, Grand Island, NY,) according to the manufacturer's
instructions. As an internal control, pooled normal human serum was used and all samples
were compared to the mean of the internal controls for each individual experiment.
All samples were measured twice, first in duplicates followed by once in singlets
and the mean of the two measurements was used for analysis.

Clinical laboratory assays

Routine laboratory testing at Department of Clinical Immunology (

Lund

University Hospital, Lund, Sweden) was used to determine levels of hemoglobin, white
blood cell count, neutrophil granulocytes, platelets, and levels of anti-DNA (Crithidia luciliae immunofluorescence test) and anti-cardiolipin titers as well as C-reactive protein
(CRP), creatinine, C1q, C3 and C4. Titers of anti-histone antibodies were determined
as previously described [18].

Statistical analysis

To determine statistical significance of difference for continuous data among three
groups or more, the Kruskal-Wallis test was used followed by a post-test using Dunn's
method for comparisons with control. To determine significant differences for nominal
data between two groups the χ2 test was used. All analyses were carried out using JMP 7 and 9 (SAS Institute, Cary,
NC, USA). Association of clinical parameters with a decreased ability to degrade NETs
was determined using conditional logistic regression with patient as strata variable
to handle repeated measurements within subject, on SAS system 9.3 (SAS).

Results

Serum samples of SLE patients were taken every two months for a median of 784 days
(mean 921 days). The duration of time for which each patient was included in the study
is displayed in Figure 1A. The disease activity as measured by SLEDAI-2K scores, recorded from the patients
at each time point, improved significantly after two years from the study inclusion
(Figure 1B).

Figure 1.Patient SLEDAI-2K decreased during the study. A. Duration of time (in years) during which the patients were included in the study.
Quantiles are shown in gray with a median of 2.1 years (784 days) and mean of 921
days (not shown). B. Individual SLEDAI-2K scores recorded from patients during the study, divided into
intervals of one year from study inclusion. Significance of difference of values compared
to the SLEDAI-2Ks during the first year was calculated using Kruskal-Wallis test followed
by Dunn's post test and is displayed as **, P < 0.01. SLEDAI-2K, Systemic Lupus Erythematosus Disease Activity Index 2000

The presence of auto antigens, such as NETs, in SLE fuels the inflammation contributing
to the disease. Therefore, we measured the ability of the patient's sera to degrade
NETs at each time point of serum collection throughout the study. To determine the
normal range of the ability to degrade NETs, sera from 77 healthy volunteers were
used (Figure 2A). A cut-off was set as previously described [8] to three standard deviations below the mean NET-degradation of the controls and thus
defined "normal" and "decreased" ability to degrade NETs. The NET-degrading abilities
were subsequently measured in all patient samples and displayed over time, starting
at study inclusion (Figure 2B). From all measurements recorded, a significant (P = 0.02) improvement was observed in the third year after study inclusion (data not
shown), but varied highly for individual patients. To quantify how common an episode
with decreased ability to degrade NETs is, the number of SLE patients whose sera never
showed decreased NET degradation was compared with sera that showed decreased degradation
at least once. We observed that 28 (41%) patients, at least once, displayed a decreased
ability to degrade NETs whereas the remaining 41 patients (59%) never displayed decreased
degradation. Out of the 28 patients whose sera at least once exhibited a decreased
ability to degrade NETs, two had a reduced ability 100% of the times (13 and 4 time
points, respectively), whereas the median frequency was at 20% (mean 34%) of all time
points (Figure 2C). Together this shows that the ability to degrade NETs may vary for each patient
over time and also that a decreased ability to degrade NETs is a rather common feature
of SLE and corresponds well to previous findings [7,8].

Figure 2.Decreased ability to degrade NETs affects 41% of the SLE patients. A-B. The ability of serum to degrade NETs compared to internal control of serum from
77 healthy controls (A) and 69 patients with SLE followed during multiple time points
(B). Dashed line indicates cut-off for "normal" and "decreased" degradation. C. The fraction of all time points for which patients, who at least once displayed
a decreased ability to degrade NETs, had a decreased NET degrading ability. Quantiles
are displayed in gray with a median of 20% and mean of 34% (not shown). NETs, neutrophil
extracellular traps; SLE, systemic lupus erythematosus

The ACR classification criteria for SLE describe cumulatively the history of a patient's
clinical manifestations since the onset of disease and give valuable information of
the clinical phenotype. To determine if there was a certain clinical subgroup of patients
who at least once displayed a decreased ability to degrade NETs compared to patients
who never did, the ACR classification criteria of the two groups were compared (Table
2). Patients who at least once exhibited a reduced NET degradation during the study
more often had a clinical history of antibodies against DNA. These patients with decreased
NET degrading ability also showed an inverse association with a history of photosensitivity,
a symptom associated with a less severe form of SLE.

We also investigated if any particular clinical manifestation, laboratory variable
or treatment preceded, co-occurred or succeeded an episode with decreased NET degradation.
To determine if a decreased degradation of NETs was associated with these variables
that occurred either two months before sampling, at sampling, or two months after
sampling, the odds ratios (OR) for the different parameters were calculated for sera
with a decreased ability to degrade NETs. The parameters were divided into three categories;
clinical manifestations derived from SLEDAI-2K, which includes mainly components of
glomerulonephritis, symptoms from skin, joints and pleuritis as well as infections
(Figure 3A), laboratory variables (Figure 3B) and medications (Figure 3C). Significant associations are also summarized in Table 3. A decreased ability to degrade NETs was associated with an increase of one point
in SLEDAI-2K score with OR 1.1 (1.0 to 1.2) before sampling, 1.2 (1.1 to 1.3) at sampling
and 1.2 (1.1 to 1.3) after sampling (Figure 3A). A decreased ability to degrade NETs was further associated with manifestations
that are considered as a part of active glomerulonephritis consisting of cellular
casts, hematuria, proteinuria, pyuria or all combined with OR 12.8 (2.3 to 67.1) for
cellular casts at the time of sampling. The OR for the presence of proteinuria before,
at the time of sampling, as well as after sampling, were 10.4 (2.49 to 43.9), 8.1
(2.9 to 23.1) and 7.4 (2.1 to 26.6), respectively. When pyuria was registered, OR
was 7.2 (2.0 to 26.0) at the time of sampling and 5.1 (1.0 to 25.2) after sampling.
Additional clinical manifestations that were not part of glomerulonephritis, but were
associated with a decreased ability to degrade NETs, were both alopecia and fever
with OR 4.8 (1.8 to 12.9) for alopecia and 5.5 (1.4 to 21.3) for fever to develop
two months after an episode with decreased ability to degrade NETs.

Figure 3.Decreased ability to degrade NETs is associated with glomerulonephritis and altered
laboratory variables. A-C. Odds ratios for co-occurrence of indicated parameters in SLE patients with a decreased
ability to degrade NETs (n: 26), divided into SLEDAI-2K and clinical manifestations (A), laboratory variables
(B) and medications (C) two months before (red), at the same time (black) or two month
after (blue) the time point with decreased ability to degrade NETs. The odds ratios
are indicated with a dot connected to the 95% confidence intervals. All values that
do not cross "1" are considered significant. NETs, neutrophil extracellular traps;
SLE, systemic lupus erythematosus; SLEDAI-2K, Systemic Lupus Erythematosus Disease
Activity Index 2000

Abnormal levels of some laboratory variables (Figure 3B) were associated with an increased OR preceding an episode with decreased ability
to degrade NETs. These factors were elevated anti-DNA antibodies with OR 2.0 (1.2
to 3.5), anti-histone antibodies 2.6 (1.0 to 6.9), the presence of antibodies against
cardiolipin 8.6 (2.0 to 37.1) and elevated CRP 3.1 (1.4 to 6.8). Furthermore, low
levels of C3 and leukocytes (leukopenia) also preceded an episode with decreased degradation
with OR 3.0 (1.3 to 7.0) and 5.8 (1.6 to 21.5), respectively. When analyzing laboratory
variables at the same time-point as the episode of decreased degradation, the following
ORs were obtained: anti-DNA antibodies 1.9 (1.3 to 2.9), anti-histone antibodies 6.6
(2.6 to 16.9) and anti-cardiolipin antibodies 4.1 (1.5 to 14.6). Reduced complement
levels were also associated with an episode of decreased degradation with OR 2.8 (1.3
to 5.7) for low C1q, 3.2 (1.6 to 6.5) for low C3 and 3.4 (1.4 to 8.3) for low levels
of C4. Leukopenia also generated an increased OR of 6.4 (1.9 to 21.6) at the time-point
of decreased degradation of NETs.

Certain medications (Figure 3C) were also more commonly administered to patients whose sera had a decreased ability
to degrade NETs. These patients were more often treated with immunosuppressants both
before and after sampling with OR of 4.5 (1.2 to 16.4) and 5.3 (1.3 to 22.2), respectively.
However, no association was seen at the time of decreased ability to degrade NETs.
Patients on hydroxychloroquine treatment displayed an inverse correlation with decreased
NET-degrading abilities with an OR of 0.2 (0.05 to 0.5) before, 0.1 (0.04 to 0.4)
after and 0.1 (0.04 to 0.3) at the time of sampling.

Discussion

In the current study we have extensively and in a truly prospective way, investigated
the ability of SLE sera to degrade NETs with the goal of determining the overall importance
of NET degradation for the disease. This was done in a longitudinal setup to reveal
temporal associations as well as potential predictive values of the NET degrading
ability. In the present study, 69 patients with SLE were followed for a median of
784 (mean 921) days with serum samples collected approximately every 60 days, generating
a total of 1,074 measurements. We found that a decreased ability to degrade NETs was
a rather common feature of SLE with 41% of the patients at least once, but commonly
more than once, displaying a reduced ability to degrade NETs.

Among the patients that never displayed a reduced ability to degrade NETs, a clinical
history of photosensitivity was more common whereas the patients who at least once
had a reduced ability to degrade NETs more often displayed a clinical history of antibodies
against DNA. Based on the cumulative classification criteria according to ACR, no
other differences could be established between the two patient groups. ACR classification
criteria reflect a cumulative clinical phenotype of SLE but not necessarily the picture
at blood sampling. Therefore, SLEDAI-2K score was used to evaluate ongoing disease
activity at the time of blood sampling. The cumulative nature of the ACR classification
criteria may also explain why no difference was observed with a history of glomerulonephritis
whereas active nephritis correlated strongly with a decreased ability to degrade NETs
and has also been established in previous studies [7,8]. Nephritis in the ACR classification criteria may reflect events, which occurred
long before the study was initiated and the NET-degrading ability at that time point
is therefore not known. Most patients were included in the study several years after
initial diagnosis.

During the study both the disease activity defined by SLEDAI-2K score as well as the
ability to degrade NETs improved for most patients. This may be due to the fact that
patients were normally recruited during active disease and an improvement would hence
be expected due to efficient medication. We have previously shown that a decreased
ability to degrade NETs is associated with a higher SLEDAI-2K score in patients, especially
reflecting patients with nephritis [8], who also normally respond well to treatment. It is interesting to note that although
no direct efforts were made to restore the ability to degrade NETs, such improvement
was achieved. This indicates that a reduced ability to degrade NETs may be a secondary
feature of SLE in a stage of active disease. Further, the results also indicate that
regular follow-ups on patients with SLE may be beneficial in reducing SLE flares.

It has previously been shown that a decreased ability to degrade NETs is strongly
associated with glomerulonephritis and the presence of antibodies directed against
DNA and histones [7,8]. By studying the ability to degrade NETs in SLE in a longitudinal setting, particular
clinical parameters could be associated with a reduced NET degrading ability (Table
3). We confirmed that patients with decreased degradation of NETs more often suffered
from manifestations of glomerulonephritis. Some manifestations of glomerulonephritis
were present both before and after the time-point with decreased NET-degradation,
whereas cellular casts, as a strong indication of glomerular damage, only occurred
during the episode with decreased degradation. Interestingly, alopecia and fever associated
strongly only two months after an episode of decreased NET degradation. This indicates
a predictive value and a potential diagnostic application but may also give insights
into possible consequences of having a reduced ability to degrade NETs and its overall
importance for pathogenesis.

Antibodies against epitopes present on NETs were further associated with a decreased
ability to degrade NETs. NETs consist of chromatin, including DNA and histones, which
are covered with antimicrobial enzymes [6]. Interestingly, elevated levels of antibodies against histone proteins were only
associated with reduced ability to degrade NETs at the same time point as the decreased
degradation occurred. Elevated and high levels (data not shown) of anti-DNA antibodies
were, however, associated with decreased degradation before, at the same time, as
well as after the episode of reduced degradation. This implies a more direct effect
or response for antibodies against histones compared to the presence of antibodies
against DNA that may have a slower turnover or response. Antibodies against DNA are
heterogeneous and their detection varies according to the employed method and may
explain the associations obtained. Notably, elevated antibody titers against cardiolipin
also resulted in an elevated OR for reduced NET degradation both before and at sampling.
The exact mechanisms for this remain to be elucidated since cardiolipin is not a known
constituent of NETs but is, however, present in the cell membrane of dead cells [20].

Low complement levels are in some cases associated with disease flare [21] and are also associated with a decreased ability to degrade NETs. Interestingly,
low C3 levels increased the OR for a reduced ability to degrade NETs within two months.
Related to that observation, elevated levels of CRP also seem to precede an episode
with a reduced ability to degrade NETs. Otherwise, low C1q and low C4 levels were
only associated with decreased NET degrading ability at the time the decrease was
observed. Patients on hydroxychloroquine treatment displayed lower ORs for an episode
with reduced ability to degrade NETs. If the antimalarial therapeutics treatment itself
has a direct effect of NET degradation is an interesting possibility; however, no
effect on NET degradation was observed when hydroxychloroquine was added directly
to serum compared to control (data not shown). The effect is, therefore, most likely
secondary or possibly due to the fact that mainly patients with less severe form of
SLE with mostly symptoms from skin and joints, excluding nephritis and presence of
antibodies against DNA, were treated with hydroxychloroquine.

The frequency of sampling in the study only allows for detection of associations within
two months (46 to 74 days) before or after a measurement and future studies with more
frequent sampling could provide further details and resolution as in how fast the
ability to degrade NETs changes over time and what parameters associate with those
changes. On the other hand, for the majority of patients the current frequency proved
narrow enough since it could take up to half a year to restore or improve a decreased
ability to degrade NETs for many patients (Figure 2B).

Attempts to treat SLE with DNase-I have been made since the 1960s, unfortunately problems
with antigenicity occurred and the trial discontinued [22]. However, new attempts were initiated with the expression of recombinant human DNase-I
[23], which was also designed to withstand the presence of potential inhibitors [24]. No clear effects were observed in an early clinical trial in 1999 [23] although animal experiments had previously shown promising results [25]. Recombinant DNase-I has, however, been developed as an FDA approved drug and is
used successfully in treating cystic fibrosis by inhalation [26]. This study reveals in which SLE patient group the treatment to restore NET degrading
ability may be most beneficial.

Conclusion

The present study confirms previously published results of manifestations that are
directly associated with a decreased ability to degrade NETs. Further, it expands,
for the first time, on previous findings in a longitudinal setting showing that a
decreased ability to degrade NETs is a common (41% of included patients) feature of
SLE. Decreased ability to degrade NETs is also associated with a history of anti-DNA
antibodies as well as active glomerulonephritis and high titers of antibodies directed
against histone proteins and DNA at the time of decreased degradation. Additional
associations indicate that a reduced ability to degrade NETs may precede fever and
alopecia. Altogether our findings support the importance of maintaining the ability
to degrade NETs in SLE and may benefit patients as a diagnostic application.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JL, MM and BG performed experiments. JL wrote the manuscript together with AMB and
AAB, who both also supervised the study. AJ maintained and collected patient data.
JAN analyzed the data statistically. All authors read and edited the manuscript.

Acknowledgements

The authors would first like to acknowledge all patients for participating in the
study. Furthermore, the authors like to acknowledge the following funding bodies:
Swedish Research Council (K2012-66X-14928-09-5), Foundations of Crafoord, Kock, King
Gustav V s 80th Anniversary, Swedish Rheumatism Association, Knut and Alice Wallenberg,
Inga-Britt and Arne Lundberg, Swedish Society of Medicine, Royal Physiographic Society
in Lund, as well as grants for clinical research (ALF and Skåne University Hospital)
and from the medical faculty at Lund University.